Probing structural elements in RNA using engineered disulfide cross-links.
Three analogs of unmodified yeast tRNAPhe, each possessing a single disulfide cross-link, have been designed and synthesized. One cross-link is between G1 and C72 in the amino acid acceptor stem, a second cross-link is in the central D region of yeast tRNAPhe between C11 and C25 and the third cross-link bridges U16 and C60 at the D loop/T loop interface. Air oxidation to form the cross-links is quantitative and analysis of the cross-linked products by native and denaturing PAGE, RNase T1 mapping, Pb(II) cleavage, UV cross-linking and thermal denaturation demonstrates that the disulfide bridges do not alter folding of the modified tRNAs relative to the parent sequence. The finding that cross-link formation between thiol-derivatized residues correlates with the position of these groups in the crystal structure of native yeast tRNAPhe and that the modifications do not significantly perturb native structure suggests that this methodology should be applicable to the study of RNA structure, conformational dynamics and folding pathways.
Maglott
E J
EJ
Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.
Glick
G D
GD
D001483:Base Sequence; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D006358:Hot Temperature; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D012331:RNA, Fungal / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D012360:RNA, Transfer, Phe / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
D003432:Cross-Linking Reagents; D004220:Disulfides; D012331:RNA, Fungal; D012360:RNA, Transfer, Phe